Study On The Role And Mechanism Of LOC339524 Gene In Inflammatory Response Of BV2 Microglial Cells Induced By Lipopolysaccharide

Objectives:LOC339524 protein is a new protein isolated and identified by mass spectrometry from human ischemic heart perfusate in our previous study.Immunohistochemical staining showed that it was expressed in brain tissue,but the role of LOC339524 was not clear.Bioinformatics suggested that LOC339524 contained a proline-rich region,so it was speculated that LOC339524 may be involved in inflammation regulation.In this experiment,we plan to study the effects of LOC339524 over-expression and silence states on inflammatory response in activated microglial cells,which would tell us the accurate role and potential mechanism of LOC339524 in inflammatory response and then provide a new idea for the regulation of microglia inflammation.Methods:1?BV2 cells in Logarithmic phase were treated by LPS(100ng/ml)for 3,6,9,and12h respectively,and then the RNA and total proteins were extracted.RT-qPCR and western blot were used to detect the expressions of TNF-?and IL-6 in different time points,and the time point that both changes were consistent would be the subsequent LPS treatment time.2?Lentiviruses were used to package previously constructed LV-TOPO-LOC339524and empty vectors.BV2 cells in logarithmic phase were infected with 1x10~8TU/ml virus titer,and the RNA and proteins were extracted from the surviving cells through being screened with the antibiotic Blasticidin.RT-qPCR and western blot were used to detect the expression of LOC339524 in the cells,which would determine whether the LOC339524over-expressed cell lines were successfully established.3?The experiment was divided into four groups:control group,LPS group,empty vector+LPS group and LOC339524 over-expression+LPS group.Cells were inoculated on6-well plates with 1×10~5 per pore.When cells were growing in logarithmic phase,the control group was replaced with serum-free DMEM/HighGlucose medium,while the others were replaced with serum-free DMEM/HighGlucose medium containing 100ng/ml LPS.12hours later,RNA and total proteins were extracted.RT-qPCR and western blot were used to detect the expressions of TNF-?,IL-6,COX2 and iNOS at the transcriptional and translational levels respectively.4?Referring to our previous research,we synthesized siLOC339524 interference fragment and NControl unrelated fragment.Cells were inoculated on 6-well plates with5×10~4 per pore.When the confluence reached about 30%,under lipo3000 mediated,the cells were transfected with 100nM/L fragments for 24,48 and 72 hour respectively.We Extracted the total proteins and then used western blot to detect LOC339524 protein expression at three time points,and selected the time point on which the silencing effect was the best as the subsequent siRNA fragments transfection time.5?The experiment was divided into four groups:control group,LPS group,NControl+LPS group and si-LOC339524+LPS group.Cells were inoculated on 6-well plates with5×10~4 per pore and were transfected with fragments when the confluence reached about30%.36 hours later,the control group was replaced with serum-free DMEM/HighGlucose medium,while the others were replaced with serum-free DMEM/HighGlucose medium containing 100ng/ml LPS.After 12 hours,RNA and total proteins were extracted.RT-qPCR and western blot were used to detect the expressions of TNF-?,IL-6,COX2 and iNOS at the transcriptional and translational levels respectively.6?The experiment was divided into control group,LPS group,empty vector+LPS group and LOC339524 over-expression+LPS group.Cells were inoculated on 6-well plates with 1×10~5 per pore.The control group was replaced with serum-free DMEM/HighGlucose medium when cells were in logarithmic phase,while the others were replaced with serum-free DMEM/HighGlucose medium containing 100ng/ml LPS.30 minutes later,total proteins were extracted,and the phosphorylation levels of p38MAPK and JNK in cells of each group were analyzed by western blot.Results:1?After stimulating BV2 cells with 100ng/ml LPS for different times,the expression of TNF-?increased,and reached a peak at 3h,then decreased,and increased again at 12h.The expression of IL-6 gradually increased with the stimulation time and reached a peak at12h.Considering that the expression changes of TNF-?and IL-6 were consistent at 12 h,12 h was selected as the subsequent LPS treatment time.2?Compared with the empty vector group,LOC339524 mRNA increased by 9.8times and protein expression level increased by 1.8 times in the LOC339524over-expression group,so the LOC339524 over-expressed BV2 cell lines were successfully established.3?After LPS stimulation,compared with the control group,the expressions of TNF-?,IL-6,COX2,and iNOS were up-regulated in the LPS group.While compared with the empty vector+LPS group,these expressions were down-regulated in the LOC339524over-expression+LPS group.4?Compared with the NControl group,LOC39524 protein expression in si-LOC339524 group was decreased by 44.2%,70.5%and 20.8%at 24?48 and 72 hour respectively.Thus,48 hour was selected as the following siRNA fragments transfection time.5?After the addition of LPS,compared with the control group,the expressions of TNF-?,IL-6,COX2 and iNOS in the LPS group were up-regulated.When compared with the NControl+LPS group,these expressions in si-LOC339524+LPS group were up-regulated.6?30 minutes later with LPS stimulation,compared with the control group,the phosphorylation levels of p38MAPK and JNK in LPS group were increased.Interestingly,compared with the empty vector+LPS group,the phosphorylation level of p38MAPK in the LOC339524 over-expression+LPS group decreased,while there was no significant change in the phosphorylation level of JNK.Conclusions:1?LOC339524 gene can inhibit LPS-induced inflammatory response in BV2microglial cells.2?The anti-inflammatory effect of LOC339524 gene is achieved by reducing the phosphorylation of p38MAPK and down-regulating the expressions of inflammatory cytokines TNF-?,IL-6 and mediators COX2 and iNOS.